Prooxidant action of desferrioxamine: Enhancement of alkaline phosphatase inactivation by interaction with ascorbate system

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 277, No. 2, March, pp. 234-240,199O

Prooxidant Action of Desferrioxamine: Enhancement Alkaline Phosphatase Inactivation by Interaction with Ascorbate System Alvaro

Mordente,’

Elisabetta

Meucci,

Giacinto

A. D. Miggiano,

and Giuseppe

of

E. Martorana

Istituto di Chimica Biologica, Universit; Cattolica de1 S. Cuore, Facoltci di Medicina e Chirurgia “Agostino Gemelli, ” Largo F. Vito 1, 00168 Rome, Ita1.y

Received June 20, 1989, and in revised form October 25,1989

Desferrioxamine (DFO) nearly doubles alkaline phosphatase oxidative inactivation by the ascorbate system. The effect is dependent on ascorbate and desferrioxamine concentrations, exhibiting in both cases a saturation mechanism. Conversion of desferrioxamine to ferrioxamine abolishes the prooxidant action. Desferrioxamine also increases ascorbate-dependent oxygen consumption and nitroblue tetrazolium reduction. Superoxide dismutase, which blocks the desferrioxamine enhancing effect on enzyme inactivation, markedly slows down nitroblue tetrazolium reduction as well as oxygen consumption by ascorbate plus desferrioxamine, while it fails to protect against the ascorbate system alone. Therefore, in the presence of desferrioxamine, the metal-catalyzed ascorbate autooxidation becomes superoxide-dependent and thus inhibitable by superoxide dismutase. Catalase, peroxidase, and ascorbate oxidase protect alkaline phosphatase from inactivation by both asaorbate and ascorbate-desferrioxamine systems. Hemin shields the enzyme from ascorbate plus DFO attack but not from ascorbate alone. In air-saturated solution, desferrioxamine seems to mediate one electron transfer from ascorbate to oxygen, generating superoxide anions, which can either trigger a Fenton reaction or produce desferal nitroxide radicals. In the absence of oxygen, ascorbate alone is ineffective, but the ascorbate plus desferrioxamine system still inactivates the enzyme; catalase, peroxidase, and ascorbate oxidase, but not superoxide dismutase, afford protection. 0 1990 Academic Press, Inc.

the treatment of haemochromatosis and other iron overload diseases (1,2). Desferrioxamine forms a stable octahedral coordination complex with Fe(II1) which cannot undergo redox cycling (3,4), thus preventing iron-catalyzed OH’ formation or lipid peroxidation (3-5). In this view, “chelation therapy” with desferal has attracted much attention as a potential treatment of several human diseases in which a “free-radical mechanism” is presumed (5,6). DFO administration, however, has been shown to lead to neurotoxic effects (7-10) and to enhance the toxicity of paraquat (11) and alloxan (12). Recently, despite conventional wisdom, Borg and Schaich (13) have hypothesized that desferrioxamine itself could display a prooxidant action producing highly reactive and potentially cytotoxic hydroxyl radicals. ESR spectroscopy studies have, moreover, detected and identified a DFO nitroxide free radical generated by the reaction of desferrioxamine with uv radiation (14), superoxide anion (l&16), and/or hydroxyl radicals (15). At this regard, investigating alkaline phosphatase (AP) posttranslational modifications by mixed function oxidation systems (17), we have surprisingly observed that DFO enhances the enzyme inactivation by the ascorbate system (18). Therefore, in view of the widespread clinical use of desferal and taking into account that ascorbic acid is often coadministered to assist mobilization of excess iron from body iron stores (19, 20), we were induced to further investigate the effects of the interaction between ascorbate and desferrioxamine. MATERIALS

Desferal (desferrioxamine, DFO ‘) is a powerful ferric iron chelator (affinity constant of 1031) currently used in ’ To whom correspondence should be addressed. * Abbreviations used: AA, ascorbate; AP, alkaline phosphatase; ADH, alcohol dehydrogenase; BSA, bovine serum albumin; DFO, des234

AND

METHODS

Materials were purchased from the following sources: lactate dehydrogenase (from rabbit muscle) and hemin from Calbiochem; L(+)-

ferrioxamine, desferal; DTPA, diethylenetriaminepentaacetic acid; FOA, ferrioxamine; EDTA, ethylenediaminetetraacetic acid; LDH, lactate dehydrogenase; NBT, nitroblue tetrazolium; O;-, superoxide anion; OH’, hydroxyl radical; SOD, superoxide dismutase. 0003.9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

DESFERRIOXAMINE TABLE Prooxidant

Action

PROOXIDANT

RESULTS

I

of Desferrioxamine

Effect of Desferrioxamine on the Inactivation Phosphatase by the Ascorbate System

Percentage of residual activity after 60 min Incubation

with

Tris AA DFO DFO + AA

235

ACTION

AP

LDH

ADH

100 67 100 34

100 90 100 66

100 70 100 55

Note. Each enzyme is incubated at 37°C in 50 mM Tris buffer, pH 7.4, with the indicated compound(s). Final concentrations are 0.1 pM AP, 0.3 /IM LDH, 0.4 GM ADH, 20 mM ascorbate, and 1 mM desferrioxamine. After 60 min, enzyme activities are assayed as described under Materials and Methods. Each result represents the mean of at least three separate experiments carried out in triplicate and in each case the values differ by no more than 5%.

ascorbic acid and EDTA disodium salt from Merck; FeCl,. 6HZ0 and CuSO, from Fluka; bovine serum albumin from Serva; CoSO, from Puratronic Johnson Matthey Chemicals; Tris, ascorbate oxidase (from squash), microbial diaphorase, and catalase (from bovine liver, superoxide dismutase free) from Boehringer; desferal (desferrioxamine B methanesulfonate) from Ciba Laboratories. Alkaline phosphatase (from bovine kidney), alcohol dehydrogenase (from yeast), xanthine oxidase (from buttermilk), peroxidase (from horseradish), superoxide dismutase (from bovine liver), cytochrome c (type VI from horse heart) and all other chemicals were obtained from Sigma. The activity of catalase (60,000 units/mg) and peroxidase (170 units/mg) was assayed as described in the Sigma catalog. Superoxide dismutase (3000 units/mg) activity was measured by the cytochrome c reduction technique (21), and xanthine oxidase (0.45 unit/mg) by the increase of absorbance at 295 nm due to urate formation. Ascorbate stock solution was prepared daily as previously reported (17). All the solutions were prepared in 50 mM Tris-HCl, pH 7.4, in highly purified water (resistivity = 18 Mohm cm) obtained through a Milli-Q water purification system (Millipore). In accordance with Bandy and Davison (22), no further attempts were made to lower the level of trace metals in the buffer solution because of the ineffectiveness in removing all transition metal ions, which were actually desired in our experiments. Atomic absorption measurements (Perkin-Elmer Model 4000) revealed that iron and copper contamination was GO.3 pM and ~0.1 PM, respectively. Ferrioxamine (Fe3+-desferal complex) was made according to Halliwell (23). Alkaline phosphatase was purified and assayed as previously described (24). Alcohol dehydrogenase and lactate dehydrogenase, used without further purification, were assayed according to Refs. (16) and (25), respectively. Anaerobic incubations were performed in a Thunberg-type cuvette, which separated the reactants until anaerobiosis was achieved by three successive cycles of evacuating the gas phase with an aspirator vacuum pump and replacing it with high-purity nitrogen or argon. Oxygen consumption was measured in 50 mM Tris-HCl buffer, pH 7.4, at 37°C in a thermostatted and sealed 3.0.ml vessel using a Clark oxygen electrode with a Yellow Springs Instrument Co. Model 53 oxygen monitor. The initial rate of ascorbate oxidation was obtained from the decrease in absorbance at 265 nm (26, 27), using a molar extinction coefficient of 14,500 Me’ cm-’ (26). Absorption changes associated with nitroblue tetrazolium (NBT) reduction were followed at 560 nm according to Ref. (23). Absorbance was measured with a Hewlett-Packard 8450 uv/vis spectrophotometer equipped with a cuvette stirring apparatus and a constant temperature cell holder. Chemiluminescence was quantified at 37°C using a 6500 Picolite luminometer (Packard). A stock solution of lucigenin (25 mM) was prepared by dissolving the dye in absolute dimethyl sulfoxide.

of Alkaline

Desferrioxamine, at concentrations slightly higher than those usually employed in in vitro experiments to inhibit OH’ formation (15), enhances alkaline phosphatase inactivation by the ascorbate system (ascorbatel O&race metals) (Table I). A direct inhibitory effect of desferrioxamine on alkaline phosphatase by chelation of structurally and/or functionally essential Zn2+ and/or Mg2+ ions can be excluded as DFO alone does not modify AP activity (Table I). In addition, other enzymes are susceptible to DFO action (see again Table I). The extent of the prooxidant effect on AP depends on DFO and ascorbate concentrations. Saturation patterns are observed either when DFO level is increased at a fixed ascorbate amount (20 mM) (Fig. 1A) or when ascorbate concentration is raised in the presence of 1 mM DFO (Fig. 1B). Superoxide dismutase, which does not protect AP from the ascorbate system alone (17), completely annuls the DFO enhancing effect (Table II), indicating the involvement of OL-. SOD protection is not a bulk protein effect since bovine serum albumin at a similar concentration fails to block DFO action (Table II). Catalase, peroxidase, and ascorbate oxidase significantly protect the enzyme from the AA plus DFO system as well as from the ascorbate system alone (17). Hemin completely

--)-/“--------I

< 4:

1.0’ 0.0

’ 0.4

’ 0.6

mol. 2.0

B 2

1.5

’ 1.2

.

1.6

JI

,,,*-g _----*------I #’ #’

t’ 8’

9,,FL----0

10 h5corbatel.

5

15 20 mm

FIG. 1. Desferrioxamine prooxidant action. Alkaline phosphatase (0.1 fiM) is incubated for 60 min at 37°C in 50 mM Tris buffer, pH 7.4, with: (A) different DFO concentrations with 20 mM AA, (B) different ascorbate concentrations with 1 mM DFO. Each point is the mean * SE of three separate experiments carried out in duplicate.

236

MORDENTE TABLE

Effect

II

of Scavenging Enzymes and Free Radical Desferrioxamine Prooxidant Action

Incubation

with

Tris DFO DFO + H202 AA Hemin + AA DFO + AA FOA FOA + AA DFO + AA sulfate DFO + SOD + AA DFO + BSA + AA DFO + catalase + AA DFO + peroxidase + AA DFO + ascorbic oxidase + AA DFO + hemin + AA DFO + formate + AA DFO + mannitol + AA DTPA DTPA + AA DTPA + DFO + AA

ET AL.

Traps

on

AP residual activity after 60 min (%o) 100 100 100 67 66 34 100 65 104 77 40 103 93 92 63 28 36 42 32 35

Note. Conditions are as in Table I. Final concentrations are 0.1 PM AP, 20 mM ascorbate, 20 mM ascorbate sulfate, 1 mM desferrioxamine, 1 mM Hz02, 1 mM ferrioxamine, 3 PM SOD, 3 FM BSA, 0.8 PM catalase, 3 PM peroxidase, 3 PM ascorbic oxidase, 3 PM hemin, 100 mM formate, 200 mM mannitol, and 10 PM DTPA. Each compound is added to the enzyme solution in the order shown in the table.

abolishes the DFO enhancing effect but does not shield alkaline phosphatase from ascorbate system attack. OK-radical traps, such as formate and mannitol, instead do not modify at any extent AP oxidative inactivation (Table II). DTPA addition reduces the degree of AP inactivation from both AA and AA plus DFO (Table II), confirming the absolute necessity of transition metal ions (even if only in trace amounts). Unlike DFO, ferrioxamine (FOA) does not enhance oxidative damage by the ascorbate system (Table II). Progressive conversion of DFO into FOA by Fe(II1) addition reduces the prooxidant effect until abolition (Fig. 2). A Hill equation bestfitted the data (n = 4.4 + 0.1; r2 = 0.997), thus suggesting that unbound hydroxylamine and carboxyl groups display a positive cooperativity and are essential for promoting DFO prooxidant activity. On the other hand, when desferrioxamine is added into the mixture containing other enzymatic and nonenzymatic hydroxyl radical generating systems, it does not enhance AP inactivation by any means (Table III). Under a nitrogen (or argon, data not shown) atmosphere, where the ascorbate system alone fails to inactivate the enzyme, the AA plus DFO system is still able to modify AP activity. Under these conditions, SOD is ineffective, whereas catalase and peroxidase as well as

0.0

0.2 0.4 0.6 0.6 [Fe3+/DFOI, ~U/1)1

FIG. 2. Influence of desferrioxamine-ferrioxamine conversion on prooxidant activity. Desferrioxamine is premixed with different concentrations of Fe3’. After 15 min, DFO or different Fe3+/DF0 complexes are incubated with alkaline phosphatase (0.1 PM) and ascorbate (20 mM) is added last. After 60 min, the enzyme activity is determined. Prooxidant activity represents the enhancement of AP inactivation by ascorbate system (0%) upon the addition of 1 mM DFO alone (100%) or different Fe3+/DF0 mixtures. Each point is the mean + SE of three separate experiments carried out in duplicate.

ascorbate oxidase still protect the enzyme by AA-DFO attack (Table IV). Desferrioxamine-Ascorbate Interaction: Polarography, Spectroscopy, and Luminescence Studies Polarographic studies show that desferrioxamine increases ascorbate-dependent oxygen consumption (Table V and Fig. 3). DFO action depends on ascorbate concentration, displaying a saturation pattern (Fig. 3). When superoxide dismutase or catalase are incubated

TABLE Desferrioxamine

Incubation

and Hydroxyl

with

Cu(I1) system Cu(I1) system + DFO (1 mM) Fe(I1) system Fe(B) system + DFO (1 mM) Co(B) system Co(B) system + DFO (1 mM) Xanthinelxanthine oxidase system Xanthinelxanthine oxidase system + DFO (1 mM) NADH/NADH oxidase system NADH/NADH oxidase system +DFO(lmM)

III Radical

Generating

Systems

AP residual activity after 60 min (%) 47 52 53 97 41 65 57 95 85 90

Note. Cu(I1) system (0.1 mM Cu2+ plus 1 mM H,O,); Fe(I1) system (0.1 mM Fe*+ plus 1 mM H,O,); Co(II) system (0.1 mM Co’+ plus 1 mM H202); xanthine/xanthine oxidase system (0.18 unit/ml xanthine oxidase/l mM xanthine plus 0.1 mM Fez’); and NADH/NADH oxidase system (1.0 unit/ml NADH oxidase/l mM NADH plus 0.1 mM Fea+). For further details see footnote to Table I.

DESFERRIOXAMINE TABLE

PROOXIDANT 2

IV

mT

Tris AA DFO DFO DFO DFO DFO DFO DFO

+ + + + + +

E!-

100 100 100 61 69 67 85 100 100

AA SOD + AA BSA + AA ascorbic oxidase + AA catalase + AA peroxidase + AA

of Desferrioxamine

.

,#$”

$’

I-: .% : 0

‘. 20

‘. 40

0, consumption (nmol/ml/min) 3.25 3.04 3.06 2.86

f 5 5 f

0.26 0.31 0.21 0.23

DFO DFO + AA DFO t SOD + AA DFO + BSA t AA DFO + catalase + AA

0 5.30 3.88 4.90 2.86

+ f + f

0.28 0.29 0.24 0.19

’ 100

, 31

DFO reaction. Superoxide anion, indeed, cannot accumulate to micromolar concentrations, as it is continuously removed by dismutation to oxygen and hydrogen peroxide or by reaction with ascorbate and/or DFO. SOD, moreover, does not affect the oxygen consumption rate by ascorbate alone (Table V). The SOD effect on the metal-catalyzed ascorbate autooxidation with or without DFO is also investigated (Table VI). DFO significantly lowers ascorbate oxidation. In the absence of desferrioxamine, SOD and BSA inhibit ascorbate autooxidation at about the same extent (Table VI) and (27). Conversely, in solutions containing desferrioxamine, SOD still slows down (50%) ascorbate oxidation, whereas BSA displays only a slight effect (5%) (Table VI). NBT reduction studies show that des-

V

on Ascorbate

AA SOD t AA BSA + AA Catalase t AA

” 80

FIG. 3. Desferrioxamine enhancement of oxygen consumption at different ascorbate concentrations. Experiments are performed as described under Materials and Methods. DFO final concentration is 10 mM. Data represent the difference between the rate of oxygen consumption in the presence of desferrioxamine minus the rate in the absence of DFO at each of the indicated ascorbate concentrations. Each point is the mean + SE of three separate experiments carried out in duplicate.

Oxygen

Consumption TABLE

Addition

” 60

[Ascot-bate]

with desferrioxamine before ascorbate addition, the DFO enhancing effect is greatly reduced (56% by SOD, 100% by catalase, only 5% by BSA). On the other hand, when catalase is added into the solution 12 min after ascorbate injection, the oxygen concentration at first increases (45%) due to the disproportionation of hydrogen peroxide. Thereafter, oxygen consumption resumes at about the same rate as when catalase is present in the reaction mixture from the beginning. No return of the consumed oxygen is detected when superoxide dismutase is added into the mixture after the start of the AA-

Effect

-

52

Y “0 c $2 lJu 58

,/f=

.

z’op

r,$ “got’

Note. Alkaline phosphatase (0.1 pM) is incubated with a N2 atmosphere at 37°C in 50 mM Tris buffer, pH 7.4, with the indicated compound(s). Final concentrations are 20 mM ascorbate, 1 mM desferrioxamine, 3 pM SOD, 3 pM BSA, 0.8 pM catalase, 3 yM peroxidase, and 3 pM ascorbic oxidase. AP activity is assayed as described under Materials and Methods.

TABLE

/*----

3-

SCH

AP residual activity after 60 min (%I

with

-*/A

C-

Prooxidant Action of Desferrioxamine under Nitrogen Atmosphere

Incubation

237

ACTION

DFO enhancing effect (%I

Protection on DFO effect (%)

Effect of Superoxide Dismutase on the Oxidation of Ascorbate Alone or Ascorbate plus Desferrioxamine

Addition

63 28 60 0

0 56 5 100

Note. Oxygen consumption experiments are performed as described under Materials and Methods. Final concentrations are 20 mM ascorbate, 10 mM desferrioxamine, 0.5 pM catalase, 0.7 pM SOD, and 0.7 pM BSA. Mean values + SE are from three independent experiments carried out in duplicate.

VI

AA oxidation (uM/min)

SOD or BSA protection (%)

AA SOD t AA BSA t AA

1.394 + 0.065 0.510 ?z0.045 0.438 k 0.023

0 63 69

DFO + AA DFO + SOD + AA DFO + BSA + AA

0.424 f 0.022 0.213 +_0.011 0.402 f 0.037

0 50 5

Note. The oxidation of ascorbate is measured at 265 nm, at 37”C, in 50 mM Tris buffer, pH 7.4. Final concentrations are 0.1 mM ascorbate, 1 mM desferrioxamine, 0.5 pM SOD, and 0.5 pM BSA. Mean values + SE are from three independent experiments carried out in duplicate. Further details are given under Materials and Methods.

238

MORDENTE TABLE

VII

Effect of Desferrioxamine on NBT Reduction Rate by Ascorbate

Addition

NBT reduction initial rate b%m/~) X lo3

AA SOD + AA BSA + AA

6.33 k 0.19 5.65 f 0.18 5.89 k 0.20

DFO DFO + AA DFO + SOD + AA DFO + BSA + AA

0 9.68 k 0.29 7.13 f 0.36 9.39 f 0.17

DFO enhancing effect (“lo)

53 26 59

Protection on DFO effect (%)

0 51 0

Note. Experiments are carried out at 37°C in 50 mM Tris, pH 7.4. Final concentrations are 3.5 mM NBT, 0.2 mM ascorbate, 1 mM desferrioxamine, 1 FM SOD, and 1 pM BSA. Mean values _+ SE are from three independent experiments performed in duplicate.

ferrioxamine also enhances the ascorbate-dependent NBT reduction (Table VII). In the presence of the chelator, superoxide dismutase markedly reduces the DFOmediated effect. On the contrary, only a slight aspecific protection is observed when SOD is added to the mixture containing only ascorbate. BSA, at the same concentration of SOD, is ineffective on NBT reduction by AA or AA plus DFO. Furthermore, desferrioxamine amplifies the maximum intensity (38%) and the integral (40%) of lucigenin luminescence induced by ascorbate (Table VIII). DISCUSSION A prooxidant effect of desferrioxamine has been first hypothesized by Borg and Schaich (13) and attributed to hydroxyl radicals produced through reduction of the ferrioxamine iron center (13). Our results, however, rule out this possibility for ascorbate, confirming what was already suggested by Schaich and Borg about the unfitness of ascorbic acid to reduce ferrioxamine (28) and demonstrating at the same time that free iron-binding sites are essential for eliciting the DFO effect. In this regard, several reports have recently described other examples of DFO prooxidant activity as well as of ferrioxamine inefficiency (29-31). Clinical studies have accordingly shown that cytotoxicity can be attained only when the dose of desferal is disproportionately high relative to that of iron (9,32,33). What kind of mechanism, then, might account for the DFO prooxidant action in the presence of the ascorbate system? The desferrioxamine prooxidant effect could be due to (a) formation of an enzyme-damaging nitroxide radical through oxidation of desferrioxamine by radical species generated during ascorbate autooxidation or (b) overproduction of hydroxyl radicals (or species of equiv-

ET AL.

alent reactivity) through a DFO-mediated cycle in which ascorbate transfers one electron to DFO which, in turn, reduces oxygen producing superoxide anion and then hydrogen peroxide. As far as the hypothesis a is concerned, ESR studies have recently shown that superoxide and hydroxyl radical-generating systems (1516) oxidize desferal to a stable nitroxide free radical, capable of inactivating alcohol dehydrogenase (16). Some oxidizing species (viz. monodehydroascorbate, hydroxyl radical, or superoxide anion), produced through a metal-catalyzed autoxidation of ascorbate, could then oxidize desferrioxamine and form an enzyme-damaging DFO radical. Nevertheless, monodehydroascorbate free radical does not seem to be involved because ascorbate oxidase and peroxidase (known to produce it through a typical one-electron oxidation of ascorbate (34, 35)) strongly protect AP. Similarly, the lack of protection by radical traps, which block ascorbate-generated OH’ in the bulk, indicates that the latter cannot trigger a DFO-dependent oxidative cycle. Furthermore, the DFO prooxidant effect is not observed in the presence of other OH’ generating systems (1,17). As far as superoxide anion is concerned, in contrast to what occurs with the ascorbate system alone (17), SOD significantly protects alkaline phosphatase by AA plus DFO, abolishing the desferal effect. In ascorbate autooxidation, the formation of superoxide anion has been discussed in some conflicting reports (27,36-38). Correspondingly, in the absence of desferrioxamine, our results are still not confirmatory and it remains difficult to determine how much of the SOD protection is due to superoxide removal and how much is caused by an aspecific complexation of metal ions by added proteins. Conversely, in the presence of desferrioxamine, SOD still slows down ascorbate oxidation whereas BSA is completely ineffective. These findings together with those from oxygen consumption, NBT reduction, and lucigenin chemiluminescence experiments strongly suggest that superoxide anion is involved in

TABLE Effect

VIII

of Desferrioxamine on Lucigenin Chemiluminescence Induced by Ascorbate Chemiluminescence

Addition Tris AA DFO DFO + AA

Maximum intensity (counts X 10e3 per 5 s) 0.510 1.235 0.548 1.550

* f f f

0.017 0.075 0.061 0.080

Integral (counts X 10-s per 10 min) 0.445 1.072 0.486 1.363

+ + + +

0.023 0.078 0.038 0.057

Note. Experiments are carried out at 37°C in 50 mM Tris, pH 7.4. Final concentrations are 1 mM lucigenin, 20 mM ascorbate, and 1 mM desferrioxamine. Mean values + SE are from two independent experiments performed in triplicate.

DESFERRIOXAMINE

PROOXIDANT

ACTION

239

protection afforded by these antioxidant enzymes. In this regard, several reducing radicals are reported to beAH’+DfNO+ Ill have as suicide substrates and/or inactivators of cataDfN(--+0;121 lase (45-47) as well as of peroxidase (48, 49). Furthermore, hemin protection is suggestive of a direct 2H+ interaction between the porphyrin ring and DFO nitroxDfNO-+0;----------+ DfNO’+H202 [31 ide radical, a known ligand of heme iron (50). NevertheDfNO’ + enzyme - - - - - - - - + oxidized enzyme [41 less, catalase and peroxidase protection, in the presence of oxygen, could also be due to impairment of the producor tion of the H202-DfNO’ complex (crypto-OH radical, a DfNO’-H202(crypto-OH’) DfNO’ + H202 ----------+ WI highly reactive OH-like species (51,52), Scheme I, reacoxidizedenzyme crypto-OH’ + enzyme -----+ WI tions [3a] and [4a]). Last, as suggested by a reviewer, the interaction between desferrioxamine and the ascorbateor metal-oxygen complex could generate a DFO nonradical Metals species such as DFO peroxide. This reactive species 0;-+H,O, -----------+ OH’+OHm+02 KW might inactivate alkaline phosphatase as well as other OH’+enzyme ---------+ oxidizedenzyme [4bl enzymes and could explain catalase and peroxidase protection. SCHEME I In contrast to ascorbate alone, the AA plus DFO system displays a strong prooxidant effect under N, or argon atmosphere, too. Even admitting some minimal proDFO prooxidant action. Superoxide could be generated through a cycle in which ascorbate (AH-) transfers one duction of oxygen active species in our anaerobic conditions, the similar extent of DFO prooxidant activelectron to desferal, forming a DFO anion radical ity in both air and nitrogen atmosphere suggests that a (DfNU2-) which in its turn reduces oxygen to 0, DFO-derived oxidizing compound is most probably the (Scheme I, reactions [l] and [ 21). At this regard, Biaglow putative enzyme-damaging species. The above considerand co-workers (39,40) and Rao et al. (41) have already reported that electron affinic drugs and carcinogens can ation should then be taken into account to explain clinical findings reporting toxicity after ascorbate suppleact as mediators or catalysts in the transfer of electrons mentation during desferal treatment (8,9,20,53,54). from ascorbate to oxygen. A similar reaction mechanism has also been proposed by Davies et al. (16) for nitroxide free radical generation by DFO-superoxide interaction. ACKNOWLEDGMENT Desferrioxamine, on the other hand, has also been deWe thank Giorgio Minotti for useful discussions andcriticism in the scribed to change the mechanism of the metal-catalyzed revision of the work. autooxidation of 6-hydroxydopamine in such a way that it becomes O;--dependent and therefore inhibitable by REFERENCES SOD (22, 42). Desferrioxamine, indeed, preventing the 1. Keberle, H. (1964) Ann. N. Y. Acad. Sci. 199,758-768. formation of ternary reductant-metal-oxygen com2. Halliwell, B., and Gutteridge, J. M. C. (1985) Mol. Aspects Med. plexes and destabilizing reduced metal-oxygen com8.89-193. plexes, forces reduction of oxygen to proceed by single 3. Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1984) electron steps with release of O;-. Superoxide anion, J. Biol. Chem. 259,3620-3624. generated by AA-DFO interaction, can either oxidize 4. Aust, S. D. (1985) Adu. Free Radical Biol. Med. 1, 103-110. DFO producing a nitroxide radical (DfNO’) as the final 5. Halliwell, B., and Gutteridge, J. M. C. (1986) Arch. Biochem. Bioenzyme-damaging species (Scheme I, reactions [3] and phys. 246,501-514. [4]) or trigger a “superoxide-driven Fenton reaction” 6. Aust, S. D., and White, B. C. (1985) Adu. Free Radical Biol. Med. with site-specific formation of OH’ radicals (not scavl,l-17. enged by radical traps) (Scheme I, reactions [3b] and 7. Arden, G. B., Wonke, B., Kennedy, C., and Huehns, E. R. (1984) [4b]). Peroxidase and catalase protection seem to imply Brit. J. Ophthnlmol. 68,873-877. involvement of hydrogen peroxide. However, in nitrogen a. Olivieri, N. F., Buncic, J. R., Chew, E., Gallant, T., Harrison, R. V., Keenan, N., Logan, W., Mitchel, D., Ricci, G., Skarf, B., or argon atmosphere, peroxides should not be produced Taylor, M., and Freedman, M. H. (1986) N. Engl. J. Med. 314, in any significant amount, thus catalase protection must 869-873. involve other chemical reactions. On the other hand, 9. Freedman, M. H., Boyden, M., Taylor, M., and Skarf, B. (1988) desferrioxamine (but not ferrioxamine) is also known to Toxicology 49,283-290. behave as a substrate for peroxidizing systems (43, 44). 10. Blake, D. R., Wyngard, P., Lunec, J., Williams, A., Good, P. A., Then, desferal and/or the radical anion produced by Crewes, S. J., Gutteridge, J. M. C., Rowley, D., Halliwell, B., CorAA-DFO interaction could act as an electron donor for nish, A., and Hider, R. C. (1985) Q. J. Med. 56,345-355. peroxidase and/or catalase and be consumed during the 11. Osherfoff, M. R., Schaich, K. M., Drew, R. T., and Borg, D. C. (1985) J. Free Radicals Biol. Med. 1,71-82. course of the reaction, explaining the almost complete In air-saturated

solution Metals AH-+DfNO---------+ DfNO+ + 0 2 -----e----e+

240

MORDENTE

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